The widespread use and applications of copper oxide nanoparticles (CuO NPs) in daily life make human exposure to these particles inevitable. This study was carried out to investigate the deteriorations in hepatic and serum biochemical parameters induced by CuO NPs in adult male mice and the potential ameliorative effect of L-arginine and quercetin, either alone or in combination. Seventy adult male mice were equally allocated into seven groups: untreated group, L-arginine, quercetin, CuO NPs, arginine + CuO NPs, quercetin + CuO NPs, and quercetin + arginine + CuO NPs. Treating mice with CuO NPs resulted in bioaccumulation of copper in the liver and consequent liver injury as typified by elevation of serum ALT activity, reduction in the synthetic ability of the liver indicated by a decrease in the hepatic arginase activity, and serum total protein content. This copper accumulation increased oxidative stress, lipid peroxidation, inflammation, and apoptosis as manifested by elevation in malondialdehyde, nitric oxide, tumor necrosis factor-α, the expression level of caspase-3 and bax quantified by qPCR, and the activity of caspase-3, in addition to the reduction of superoxide dismutase activity. It also resulted in severe DNA fragmentation as assessed by Comet assay and significant pathological changes in the liver architecture. The study proved the efficiency of quercetin and L-arginine in mitigating CuO NPs-induced sub-chronic liver toxicity due to their antioxidant, anti-inflammatory, and anti-apoptotic properties; ability to inhibit DNA damage; and the potential as good metal chelators. The results of histopathological analysis confirmed the biochemical and molecular studies.